Previous studies (Pharmacologist 20:180,1978; Fed. Proc. 38:683,1979) have shown that phenylbutazone (150 mg/kg, i.p.) causes massive hepatic necrosis in rats, especially in those that had been pretreated with both phenobarbital and diethyl maleate. Also the incidence and severity of phenylbutazone-induced hepatotoxicity are correlated with changes in the in vivo covalent binding of chemically reactive metabolites of phenylbutazone after various pretreatments. In vitro studies indicated that covalent binding of phenylbutazone to microsomal protein requires oxygen and NADPH as a cofactor and is inhibited by a CO-O2 (4:1) atmosphere, and nitrogen. Thus cytochrome P-450 appears to catalyze the formation of these reactive metabolites. The structures of these chemically reactive metabolites remain to be elucidated.